水通道蛋白(aquaporins, AQPs)是一群執行水分子通透的細胞膜蛋白。此外，有些AQPs也被發現具有二氧化碳、甘油、氨與尿素的通透性。因此AQPs 依其功能又區分成三亞群，分別為aquaporins, aquaammoniaporins, 與 aquaglyceroporins三群。在哺乳類研究發現，AQP1缺失的紅血球會降低二氧化碳通透性。最近研究將斑馬魚(Danio rerio) aqp1a表現於蛙卵會增加細胞膜對二氧化碳通透性。然而，目前仍沒有活體的實驗證實AQPs在動物體內參與二氧化碳(carbon dioxide, CO2)通透。本研究利用斑馬魚仔魚為模式動物，探討aqp1a在仔魚表皮細胞上的分佈與功能。將1 % CO2馴養一週的仔魚以real-time PCR分析，結果顯示aqp1a mRNA表現量增加。利用原位雜交與抗體染色標定，發現aqp1a大量表現於卵黃囊表皮上的H+-pump-rich cell與Na+ -pump-rich cell，其他表皮細胞則有少量的表現。利用morpholino knockdown弱化aqp1a蛋白的表現再利用離子選擇電極技術(SIET)分析碳酸排放，發現aqp1a基因弱化的仔魚碳酸的排放減少，顯示aqp1a在胚胎體表細胞扮演CO2通透的功能。

Aquaporins (AQPs, water channels) are integral membrane proteins that facilitate water transport across cell membrane. However, some member of AQPs was also found to facilitate transport of carbon dioxide (CO2), glycerol, ammonia, and urea. According to their properties, AQPs were divided into three functional groups: aquaporins, aquaammoniaporins, and aquaglyceroporins. In mammalian studies, AQP1-deficient erythrocytes were found to decrease permeability to CO2. In vitro studies by expressing mammalian AQP1 or zebrafish aqp1a in Xenopus oocytes showed that AQP1 is able to facilitate CO2 diffusion. However, in vivo study is still lacking to demonstrate AQP1 is involved in CO2 transport in mammals or other vertebrates. In this study, we used zebrafish larvae as an in vivo model to investigate the function of aqp1a. Results showed that aqp1a mRNA in larvae was induced by hypercapnia (1% CO2) treatment for 7 days. In situ hybridization of aqp1a showed that it was highly expressed in H+-pump-rich cells (HRCs) and Na+ pump-rich cells (NaRCs) and slightly expressed in Na+/Cl- cotrasporter (NCC) cells and keratinocytes of larval skin. Using morpholino knockdown technique to suppress the protein expression of aqp1a and scanning ion-selective electrode technique (SIET) to analyze carbonic acid formation at the surface of specific skin cells, this study demonstrates that aqp1a plays a critical role in CO2 diffusion across the skin of zebrafish larvae.

Agre, P., (2006). The aquaporin water channels. Proc. Am. Thorac. Soc. 3, 5–13.
An, K.W., Kim, N.N. and Choi, C.Y., (2008). Cloning and expression of aquaporin 1 and arginine vasotocin receptor mRNA from the black porgy, Acanthopagrus schlegeli: effect of freshwater acclimation. Fish. Physiol. Biochem. 34, 185-194.
Aoki, M., Kaneko, T., Katoh, F., Hasegawa, S., Tsutsui, N. and Aida, K., (2003). Intestinal water absorption through aquaporin 1 expressed in the apical membrane of mucosal epithelial cells in seawater-adapted Japanese eel. J.Exp. Biol. 206, 3495-3505.
Bobe, J., Montfort, J., Nguyen, T. and Fostier, A., (2006). Identification of new participants in the rainbow trout (Oncorhynchus mykiss) oocyte maturation and ovulation processes using cDNA microarrays. Reprod. Biol. Endocr. 4, 39.
Brunelli, E., Mauceri, A., Salvatore, F., Giannetto, A., Maisano, M. and Tripepi, S., (2010). Localization of aquaporin 1 and 3 in the gills of the rainbow wrasse Coris julis. Acta. Histochem. 112(3), 251-258.
Chen, L.M., Zhao, J., Musa-Aziz, R., Pelletier, M.F., Drummond, I.A. and Boron, W.f., (2010). Cloning and characterization of a zebrafish homologue of human AQP1: a bifunctional water and gas channel. Am. J. Physiol. Regul. Integr. Comp. Physiol. 299, 1163-1174.
Cutler, C.P. and Cramb, G., (2002). Branchial expression of an aquaporin 3(AQP-3) homologue is downregulated in the European eel Anguilla anguilla following seawater acclimation. J. Exp. Biol. 205, 2643-2651.
Cutler, C.P., Martinez, A.S. and Cramb, G., (2007). The role of aquaporin 3 in teleost fish. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 148, 82-91.
Deane, E.E. and Woo, N.Y.S., (2006). Tissue distribution, effects of salinity acclimation and ontogeny of aquaporin 3 in the marine teleost, silver sea bream (Sparus sarba). Mar. Biotechnol. 8, 663-671.
Denker, B.M., Smith, B.L., Kuhajda, F.P. and Agre, P., (1988). Identification, purification, and partial characterization of a novel Mr 28,000 integral membrane protein from erythrocytes and renal tubules. J. Biol. Chem. 263, 15634-15642.
Endeward, V., Musa-Aziz, R., Cooper, G.J., Chen, L.M., Pelletier, M.F., Virkki, L.V., Supuran, C.T., King, L.S., Boron, W.F. and Gros, G., (2006). Evidence that aquaporin 1 is a major pathway for CO2 transport across the human erythrocyte membrane. FASEB J. 20, 1974-1981.
Evans, D. H., (2008). Teleost fish osmoregulation: what have we learned since August Krogh, Homer Smith, and Ancel Keys. Am. J. Physiol. Regul. Integr. Comp. Physiol. 295, R704-713.
Evans, D. H., Piermarini, P. M. and Choe, K. P., (2005). The multifunctional fish gill: dominant site of gas exchange, osmoregulation, acid-base regulation, and excretion of nitrogenous waste. Physiol. Rev. 85, 97-177.
Forster, R. E., Gros, G., Lin, L., Ono, Y. and Wunder, M., (1998). The effect of 4,4'-diisothiocyanato-stilbene-2,2'-disulfonate on CO2 permeability of the red blood cell membrane. Proc. Natl. Acad. Sci. U S A. 95, 15815-15820.
Giffard-Mena, I., Boulo, V., Aujoulat, F., Fowden, H., Castille, R., Charmantier, G. and Cramb, G., (2007). Aquaporin molecular characterization in the sea-bass (Dicentrarchus labrax): the effect of salinity on AQP1 and AQP3 expression.Comp. Biochem. Physiol. A Mol. Integr. Physiol. 148, 430-444.
Hemptinne, A. and Huguenin, F.,(1984). The influence of muscle respiration and glycolysis on surface and intracellular pH in fibres of the rat soleus. J. Physiol. 347, 581-592.
Horng, J.L., Lin, L.Y. and Hwang, P.P., (2009). Functional regulation of H+-ATPase-rich cells in zebrafish embryos acclimated to an acidic environment. Am J Physiol Cell Physiol. 296(4), C682-692
Horng, J.L., Lin, L.Y., Huang, C.J., Katoh, F., Kaneko, T. and Hwang, P.P.,(2007). Knockdown of V-ATPase subunit A (atp6v1a) impairs acid secretion and ion balance in zebrafish (Danio rerio). Am J Physiol Regul Integr Comp Physiol. 292(5), R2068-2076.
Hwang, P. P., (2009). Ion uptake and acid secretion in zebrafish (Danio rerio). J. Exp. Biol. 212, 1745-1752.
Hwang, P. P. and Lee, T. H., (2007). New insights into fish ion regulation and mitochondrion-rich cells. Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 148, 479-497.
Hwang, P.P., Lee, T.H. and Lin, L.Y., (2011). Ion regulation in fish gills: recent progress in the cellular and molecular mechanisms. Am. J. Physiol. Regul. Integr. Comp. Physiol. (Epub ahead of print)
Ishibashi, K., Sasaki, S., Fushimi, K., Uchida, S., Kuwahara, M., Saito, H., Furukawa, T., Nakajima, K., Yamaguchi, Y. and Gojobori, T., (1994). Molecularcloning and expression of a member of the aquaporin family with permeability to glycerol and urea in addition to water expressed at the basolateral membrane of kidney collecting duct cells. Proc. Natl. Acad. Sci. U S A. 91(14), 6269-6273.
Lignot, J.H., Cutler, C.P., Hazon, N. and Cramb, G., (2002). Immunolocalisation of aquaporin 3 in the gill and the gastrointestinal tract of the European eel Anguilla anguilla. J. Exp. Biol. 205, 2653-2663.
Lin, L. Y., Horng, J. L., Kunkel, J. G. and Hwang, P. P., (2006). Proton pump-rich cell secretes acid in skin of zebrafish larvae. Am. J. Physiol. Cell. Physiol. 290, C371-378.
Lin, T.Y., Liao, B.K., Horng, J.L., Yan, J.J., Hsiao, C.D. and Hwang, P.P., (2008). Carbonic anhydrase 2-like a and 15a are involved in acid-base regulation and Na+
uptake in zebrafish H+-ATPase-rich cells. Am. J. Physiol. Cell. Physiol. 294(5),
C1250-1260.
Litman, T., Søgaard, R. and Zeuthen, T., (2009). Ammonia and urea permeability of mammalian aquaporins. Handb. Exp. Pharmacol. 190, 327-358.
Martínez, A.S., Cutler, C.P., Wilson, G.D., Phillips, C., Hazon, N. and Cramb, G. (2005). Regulation of expression of two aquaporin homologs in the intestine of the European eel: effects of seawater acclimation and cortisol treatment. Am. J. Physiol. Regul. Integr. Comp. Physiol. 288, 1733-1743.
McLamore, E.S., Porterfield, D.M. and Banks, M.K., (2009). Non-invasive self-referencing electrochemical sensors for quantifying real-time biofilm analyte flux. Biotechnol. Bioeng. 102(3), 791-799.
Musa-Aziz, R., Chen, L.M., Pelletier, M.F. and Boron, W.F., (2009a). Relative CO2/NH3 selectivities of AQP1, AQP4, AQP5, AmtB, and RhAG. Proc. Natl. Acad. Sci. U S A. 106(13), 5406-5411.
Nakhoul, N.L., Davis, B.A., Romero, M.F. and Boron, W.F., (1998). Effect of expressing the water channel aquaporin-1 on the CO2 permeability of Xenopus oocytes. Am. J. Physiol. 274, 543-548.
Nawata, C.M., Hirose, S., Nakada, T., Wood, C.M. and Kato, A., (2010). Rh
glycoprotein expression is modulated in pufferfish (Takifugu rubripes) during
high environmental ammonia exposure. J. Exp. Biol. 213(Pt 18), 3150-3160.
Perry, S.F., Braun, M.H., Noland, M., Dawdy, J. and Walsh, P.J., (2010). Do zebrafish Rh proteins act as dual ammonia-CO2 channels? J. Exp. Zool. A Ecol Genet Physiol. 313(9), 618-621.
Prasad, G.V., Coury, L.A., Finn, F. and Zeidel, M.L., (1998). Reconstituted aquaporin 1 water channels transport CO2 across membranes. J. Biol. Chem. 273(50), 33123-33126.
Preston, G.M., Carroll, T.P., Guggino, W.B. and Agre, P., (1988). Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein. Science. 256, 385-387.
Purkerson, J.M. and Schwartz, G.J., (2007). The role of carbonic anhydrases in renal physiology. Kidney Int. 71, 103-115.
Raldúa, D., Otero, D., Fabra, M. and Cerdà, J., (2008). Differential localization and regulation of two aquaporin-1 homologs in the intestinal epithelia of the marine teleost Sparus aurata. Am. J. Physiol. Regul. Integr. Comp. Physiol. 294, 993- 1003.
Rojek, A., Praetorius, J., Frøkiaer, J., Nielsen, S. and Fenton, R.A., (2008). A current view of the mammalian aquaglyceroporins. Annu. Rev. Physiol.70, 301-327.
Shih, T. H., Horng, J. L., Hwang, P. P. and Lin, L. Y., (2008). Ammonia excretion by the skin of zebrafish (Danio rerio) larvae. Am. J. Physiol. Cell. Physiol. 295, C1625-1632.
Smith, P.J., Hammar, K., Porterfield, D.M., Sanger, R.H., Trimarchi, J.R., (1999). Self-referencing, non-invasive, ion selective electrode for single cell detection of trans-plasma membrane calcium flux. Microsc. Res. Tech. 46(6), 398-417.
Tingaud-Sequeira, A., Calusinska, M., Finn, R.N., Chauvigné, F., Lozano, J. and Cerdà, J., (2010). The zebrafish genome encodes the largest vertebrate repertoire of functional aquaporins with dual paralogy and substrate specificities similar to mammals. BMC Evol. Biol. 10, 38.
Tipsmark, C.K., Sørensen, K.J. and Madsen, S.S., (2009). Aquaporin expression dynamics in osmoregulatory tissues of Atlantic salmon during smoltification and seawater acclimation. J. Exp. Biol. 213, 368-379.
Tse, W. K. F., Au, D. W. T. and Wong, C. K. C., (2006). Characterization of ion channel and transporter mRNA expressions in isolated gill chloride and pavement cells of seawater acclimating eels. Biochem. Biophys. Res. Commun. 346, 1181-1190.
Uehlein, N., Lovisolo, C., Siefritz, F., Kaldenhoff, R., (2003). The tobacco aquaporin NtAQP1 is a membrane CO2 pore with physiological functions. Nature. 425(6959), 734-737.
Verkman, A.S., (2009). Knock-out models reveal new aquaporin functions. Handb. Exp. Pharmacol. 190, 359-381.
Watanabe, S., Hirano, T., Grau, E.G. and Kaneko, T., (2008). Osmosensitivity of prolactin cells is enhanced by the water channel aquaporin-3 in a euryhaline Mozambique tilapia (Oreochromis mossambicus). Am. J. Physiol. Regul. Integr. Comp. Physiol. 296, 446-453.
Watanabe, S., Kaneko, T. and Aida, K., (2005). Aquaporin-3 expressed in the
basolateral membrane of gill chloride cells in Mozambique tilapia Oreochromis
mossambicus adapted to freshwater and seawater. J. Exp. Biol. 208, 2673-2682.